Introduction

Mitoxantrone (1,4-dihydroxy-5,8-bis [[2-[2-hydroxyethyl)amino]ethyl]-9,10-anthracenedione) is an antitumor hydroquinone that is considered to be a synthetic analog of doxorubicin.¹ Doxorubicin is an anthracycline with a broad range of therapeutic activity. Its major toxicities include myelosuppression and cardiac toxicity.² Mitoxantrone was synthesized in an attempt to develop anthracycline analogs with less cardiac toxicity. Mitoxantrone inhibits DNA and RNA synthesis, intercalates DNA, and causes single- and double-stranded DNA breaks.³ Like doxorubicin, mitoxantrone’s cytotoxic effects also include topoisomerase-II-dependent damage.³ However, unlike doxorubicin, mitoxantrone is less likely to undergo oxidation reduction reactions and form free radicals.⁴ A study using healthy research dogs showed that mitoxantrone does not result in cardiotoxicity in dogs and as a result, it may be used in place of doxorubicin in patients for whom doxorubicin chemotherapy is considered contraindicated or for those patients who have reached the maximal cumulative dose of doxorubicin.^1,4

Mitoxantrone has shown activity against a variety of canine neoplasms including lymphoma, squamous cell carcinoma, and transitional cell carcinoma.^5–8 In human medicine, mitoxantrone has antineoplastic activity in metastatic breast cancer, liver cancer, non-Hodgkin’s lymphoma, and acute lymphoid leukemia, and is approved for treatment of advanced hormone-refractory prostate cancer.²

Adverse events (AEs) associated with mitoxantrone chemotherapy in dogs include vomiting, diarrhea, lethargy, and anorexia. The established dose-limiting AE of mitoxantrone in the dog is neutropenia.^9,10 Neutropenia resulting in sepsis is potentially life threatening, and it has been shown that dogs with lower body weights are at increased risk for chemotherapy-induced sepsis.¹¹ The current use of body surface area to calculate the dose of chemotherapy drugs may result in overdosing of smaller animals.¹² In a study evaluating the toxicoses associated with administration of mitoxantrone to dogs with malignant tumors, the AEs associated with the administration of mitoxantrone at a dosage of 5 mg/m² were significant enough to preclude the use of higher doses. The most common AEs were vomiting, diarrhea, anorexia, and sepsis as a result of myelosuppression.⁹ Another study evaluated toxicoses associated with dose escalation of mitoxantrone in dogs with malignant tumors. The most common toxicoses were vomiting, diarrhea, lethargy, and sepsis. Two dogs died of complications attributable to mitoxantrone administration. It was concluded that the morbidity of adverse effects at a dosage of 6.5 mg/m² precluded the use of higher doses and suggested that mitoxantrone should be administered at a dosage of 6.0 mg/m².¹⁰

The purpose of this retrospective study was to investigate the incidence of neutropenia in dogs being treated with their first dose of single-agent mitoxantrone chemotherapy at 5 mg/m². The hypothesis was that small canine patients are more likely than large dogs to experience grade 3 or 4 neutropenia following treatment with mitoxantrone.

Materials and Methods

Adverse Event Assessment

Hematologic AEs were assessed and scored based on the toxicity criteria of the Veterinary Cooperative Oncology Group (Table 1).¹³ Grade 1 neutropenia was classified as a neutrophil count less than the lower limit of normal at each reference lab, grade 2 as 1000–1499 cells/uL, grade 3 as 500–999 cells/uL, and grade 4 as <500 cells/uL. Although all grades of neutropenia were recorded, grades 3 and 4 neutropenia were considered most likely to result in moderate to severe toxicity.¹

TABLE 1 Veterinary Cooperative Oncology Group Definition of Hematologic Adverse Events¹⁰

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Results

A total of 37 dogs met the inclusion criteria, of whom 25 were male (24 castrated) and 12 were spayed females. The median age was 10 yr. Twenty-three breeds were represented, including mixed-breed dog (n = 8), Staffordshire bull terrier (n = 3), Scottish terrier (n = 2), West Highland white terrier (n = 3), Labrador retriever (n = 2), Shetland sheepdog (n = 2), Yorkshire terrier (n = 1), American Eskimo (n = 1), bichon frise (n = 1), beagle (n = 1), Kerry blue terrier (n = 1), poodle (n = 1), Dalmatian (n = 1), basenji (n = 1), Australian shepherd (n = 1), basset hound (n = 1), Kuvasz (n = 1), whippet (n = 1), Weimaraner (n = 1), Doberman pinscher (n = 1), mastiff (n = 1), border collie (n = 1), and Newfoundland (n = 1). Eight dogs weighed ≤10 kg, seven dogs weighed 10.1 to ≤15 kg, and twenty-two dogs had a body weight >15 kg. The median body weight was 23.4 kg (range: 3.55–62 kg). Tumor histologies included transitional cell carcinoma (n = 29), prostatic carcinoma (n = 2), mammary carcinoma (n = 1), hemangiosarcoma (n = 3), metastatic carcinoma of unknown primary (n = 1), and anal sac adenocarcinoma (n = 1). Four dogs had received previous antineoplastic therapy including doxorubicin^f, epirubicin^g, and toceranib phosphate^h. Concurrent medications included nonsteroidal anti-inflammatory medications (meloxicamⁱ [n = 20] piroxicam^j [n = 1]), analgesics (gabapentin^k [n = 1] and tramadol^l [n = 2]), and anti-inflammatories (trimeprazine tartrate with prednisolone^m [n = 1]).

Adverse Event Assessment

The Veterinary Cooperative Oncology Group grading scheme for hematologic AEs was used to evaluate the complete blood count on day 7 following mitoxantrone administration. Overall, 5 dogs (14%) experienced grade 4 neutropenia (median: 330 cells/uL; range: 140–400 cells/uL), 8 (21%) experienced grade 3 neutropenia (median: 750 cells/uL; range: 640–940 cells/uL), no dogs experienced grade 2 neutropenia, and 13 (35%) dogs experienced grade 1 neutropenia on day 7 following mitoxantrone chemotherapy (median: 2200 cells/uL; range: 1500–2700 cells/uL). Of the eight dogs weighing ≤10 kg, all dogs experienced grade 3 or 4 neutropenia; four experienced grade 3 neutropenia (median: 750 cells/uL; range: 660–860 cells/uL) and four experienced grade 4 neutropenia (median: 250 cells/uL; range: 140–390 cells/uL). Of the seven dogs with body weights between 10.1 and ≤15 kg, three dogs developed grade 3 or 4 neutropenia; two experienced grade 3 neutropenia (median: 750 cells/uL; range: 700–800 cells/uL) and one experienced grade 4 neutropenia (400 cells/uL). Of the 22 weighing >15 kg, 2 had grade 3 neutropenia (median: 790 cells/uL; range: 640–940 cells/uL), and no grade 4 neutropenia was reported (Table 2). No other hematologic AEs were noted.

TABLE 2 Frequency of Neutropenia Based on Patient Weight

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Dogs ≤10 kg body weight were significantly more likely to develop grade 3 or 4 neutropenia (5.8 RR; 95% confidence interval [CI], 2.6–12.9; P < .0001) than dogs >10 kg. Dogs ≤15 kg body weight were significantly more likely to develop grade 3 or 4 neutropenia (8.1 RR; 95% CI, 2.1–31.3; P < .0001) than dogs >15 kg. Dogs ≤10 kg body weight were also significantly more likely to develop grade 3 or 4 neutropenia (2.3 RR; 95% CI, 1.0–5.5; P = .026) than dogs weighing 10.1 to ≤15 kg.

Overall, 6 of the 13 (46%) patients who developed grade 3 or 4 neutropenia were hospitalized. Four patients weighing ≤10 kg experiencing grade 4 neutropenia were hospitalized with clinical signs related to neutropenia. All four patients were pyrexic and lethargic, but no gastrointestinal toxicity was reported, and they were treated with parenteral administration of broad-spectrum antibiotics (enrofloxacinⁿ and ampicillin^o) and intravenous fluid therapy. Two patients experiencing grade 3 neutropenia were hospitalized for pyrexia and lethargy and treated with parenteral administration of broad-spectrum antibiotics (enrofloxacinⁿ and ampicillin^o) and intravenous fluid therapy (one patient had a body weight ≤10 kg, and one patient had a body weight 10.1 to ≤15kg). The median duration of hospitalization was 1.5 days (range: 1–3 days).

Seven patients experiencing grade 3 or 4 neutropenia were not hospitalized. One patient weighing >10 kg experiencing grade 4 neutropenia was treated with oral broad-spectrum antibiotics but was asymptomatic and was treated on an outpatient basis. Three patients with grade 3 neutropenia were asymptomatic (all with body weights >10 kg) and were treated with prophylactic oral broad-spectrum antibiotics, and one patient weighing ≤10 kg was lethargic and received oral broad-spectrum antibiotics. Information regarding clinical signs related to neutropenia was unavailable for two patients; however, the patients were not hospitalized and did not receive antibiotic therapy.

Among the patients experiencing neutropenia, one Shetland sheepdog (8.88 kg) experienced grade 4 neutropenia. This patient was pyrexic and lethargic and was treated with parenteral administration of broad-spectrum antibiotics (enrofloxacinⁿ and ampicillin^o) and intravenous fluid therapy, whereas the other Shetland sheepdog (14.3 kg) had grade 1 neutropenia with no clinical signs. The Australian shepherd (26.3 kg) experienced grade 1 neutropenia and was asymptomatic.

Dogs weighing ≤10 kg (n = 8) had significantly lower neutrophil counts at day 7 than dogs weighing >10 kg (n = 29; P = .000002; mean neutrophil counts 506.3 cells/uL versus 2963.8 cells/uL). Similarly, there was a significant difference in neutrophil counts at day 7 between dogs weighing ≤10 kg (n = 8) and >15 kg (n = 22; P = .003; mean neutrophil counts 506.3 cells/uL versus 3341.4 cells/uL). The difference was not significant between other groups in the one-way ANOVA. No statistically significant differences were found in neutrophil counts at day 0 between any of the groups evaluated (P > .05). Furthermore, a significant (P = .002) but weak (r = 0.499) positive correlation was observed between body weight and neutrophil counts at day 7.

Discussion

In the current study, all dogs weighing ≤10 kg experienced grade 3 or higher neutropenia on day 7 following mitoxantrone chemotherapy, two (28%) dogs weighing 10.1 to ≤15 kg experienced grade 3 neutropenia, and one (14%) dog experienced grade 4 neutropenia. Only two (9%) dogs weighing >15 kg experienced grade 3 neutropenia and none experienced grade 4 neutropenia. These data show that dogs weighing ≤10 and ≤15 kg experience a 5.8 and 8.1 RR of developing grade 3 or 4 neutropenia, respectively, compared with dogs >15 kg. Although the risk of neutropenia is increased in dogs 10.1 to ≤15 kg, only one patient had asymptomatic grade 4 neutropenia and one had grade 3 neutropenia that required hospitalization. In contrast, all four patients weighing ≤10 kg experiencing grade 4 neutropenia were hospitalized for clinical signs, and one of four patients weighing ≤10 kg experiencing grade 3 neutropenia required hospitalization. Based on the results of the current study, the initial starting dose of mitoxantrone should likely be lower for dogs weighing ≤10 kg, and clinicians should be aware of the increased risk of neutropenia in patients 10.1 to ≤15 kg.

At our institution, the initial dose of mitoxantrone administered to dogs is 5 mg/m². This dose is used because a previous study showed that doses >5 mg/m² resulted in severe neutropenia in four of five clinically normal dogs.¹⁴ One study evaluated AEs associated with mitoxantrone administration to dogs with malignant tumors using doses of 2.5–5 mg/m². In that study, 12 of 129 dogs developed sepsis secondary to neutropenia. As a result of the AEs seen with mitoxantrone administration at 5 mg/m², no higher doses were administered to dogs in that study.⁹ In contrast, another study evaluated AEs associated with escalating doses of mitoxantrone.¹⁰ In that study, 23 dogs had received mitoxantrone at a dosage of 6.5 mg/m², and the median neutrophil count on day 7 following treatment was 2800 cells/uL. Six of those dogs had neutrophil counts <1000 cells/uL, and three dogs had neutrophil counts <500 cells/uL. In the same study, the median neutrophil count of the 11 dogs given mitoxantrone at a dosage of 6.0 mg/m² was 3800 cells/uL. Two of those dogs developed neutrophil counts <1000 cells/uL. Finally, the median neutrophil count of the dogs given mitoxantrone at a dosage of 5.5 mg/m² was 4500 cells/uL, with no dogs having a neutrophil count <1500 cells/uL. Four of the eight dogs in that study who developed grade 3 or 4 neutropenia experienced clinical signs of sepsis as a result of their neutropenia, with neutrophil counts <1500 cells/uL. Two dogs in the study died as a result of mitoxantrone treatment, and both had been given doses of 6.5 mg/m². It is important to note that the median body weight of the dogs in that study was 29 kg (range: 7.73–55.9 kg). As a result, it is not possible to determine whether the incidence of neutropenia would have been greater at all doses administered if there was a greater proportion of small dogs.

Many chemotherapeutic drugs are dosed based on body surface area rather than body weight. Body surface area is thought to correlate better with basal metabolic rate, blood volume, cardiac output, and pharmacokinetic drug disposition than body weight.¹⁵ However, a number of studies have demonstrated that dosing chemotherapeutic drugs based on body surface area may result in overdosing in small dogs.^11,16,17 When doxorubicin was administered to 17 normal dogs, 5 of 9 dogs weighing <10 kg developed grade 3 or 4 neutropenia after receiving doxorubicin at a dosage of 30 mg/m². In contrast, myelosuppression was not observed in small dogs receiving doxorubicin at a dosage of 1 mg/kg.¹⁷ Similarly, greater toxicity was noted in dogs weighing <14 kg following administration of melphalan to patients with spontaneously occurring neoplasia, with seven of eight small dogs developing grade 3 or 4 neutropenia. In contrast, only 3 of 13 dogs weighing >14 kg developed severe myelosuppression.¹⁶ In the current study, all dogs weighing ≤10 kg developed grade 3 or 4 neutropenia, in contrast to only 43% of dogs weighing 10.1 to ≤15 kg and 9% of the dogs weighing >15 kg. Furthermore, a study evaluating risk factors for the development of sepsis in dogs receiving chemotherapy showed that septic cases were found to weigh less than control dogs.¹¹ These finding support the results of the current study, in which 6 of the 13 (46%) patients who developed grade 3 or 4 neutropenia were hospitalized for clinical signs related to neutropenia. Five of those patients had body weights ≤10 kg, whereas only one patient with a body weight 10.1 to ≤15 kg required hospitalization for clinical signs related to neutropenia.

Four dogs in this study had received other chemotherapy treatment <1 mo prior to being treated with mitoxantrone. Of those dogs, two had body weights ≤10 kg, one weighed 28 kg, and one weighed 51 kg. None of these dogs experienced neutropenia with previous chemotherapy. Only the two dogs weighing ≤10 kg developed neutropenia following mitoxantrone chemotherapy. The impact of previous chemotherapy on the development of neutropenia following mitoxantrone treatment in this study is unknown. However, a previous study did not find any association between myelosuppression following mitoxantrone chemotherapy and previous chemotherapy treatment.⁹

The limitations of this study include its retrospective nature. It was not always possible to determine from the medical records if the patients in this study experienced clinical signs related to myelosuppression. However, the purpose of this study was to investigate the incidence of neutropenia in patients treated with single-agent mitoxantrone, not clinical outcome.

Mutation analysis for multidrug sensitivity was not evaluated for the dogs in this study. This represents another limitation of the current study, as MDR1 mutation is associated with increased susceptibility to many adverse drug reactions (including mitoxantrone) in several breeds, including Shetland sheepdogs and Australian shepherds.¹⁸ The study reported here included two Shetland sheepdogs and one Australian shepherd. One Shetland sheepdog experienced grade 4 neutropenia and was hospitalized and the other had grade 1 neutropenia with no clinical signs. The Australian shepherd experienced grade 1 neutropenia and was asymptomatic. Due to the lack of testing for the MDR1 mutation in these patients, it is not possible to determine if the neutropenia AE was associated with MDR status.

Conclusion

This study supports the findings of previous studies that showed that small dogs are at greater risk of myelosuppression following administration of certain chemotherapeutic agents. In this study, 100% of dogs weighing ≤10 kg developed grade 3 or 4 neutropenia.

Dogs weighing ≤10 and ≤15 kg experienced a 5.8 and 8.1 RR of developing grade 3 or 4 neutropenia, respectively, compared with larger dogs. However, dogs weighing 10.1 to ≤15 kg were more likely to experience asymptomatic neutropenia compared with dogs ≤10 kg. These results suggest that in dogs weighing ≤10 kg, a dose reduction should be considered for the initial dose of mitoxantrone chemotherapy, and clinicians should be aware of the increased risk of neutropenia in patients weighing 10.1 to ≤15 kg.